Cholesterol implications in plasmid DNA electrotransfer: Evidence for the involvement of endocytotic pathways

The delivery of therapeutic molecules such as plasmid DNA in cells and tissues by means of electric fields holds great promise for anticancer treatment. To allow for their therapeutic action, the molecules have first to traverse the cell membrane. The mechanisms by which the electrotransferred pDNA interacts with and crosses the plasma membrane are not yet fully explained. The aim of this study is to unravel the role of cholesterol during gene electrotransfer in cells. We performed cholesterol depletion experiments and measured its effects on various steps of the electroporation process. The first two steps consisting of electropermeabilization of the plasma membrane and of pDNA interaction with it were not affected by cholesterol depletion. In contrast, gene expression decreased. Colocalization studies with endocytotic markers showed that pDNA is endocytosed with concomitant clathrin- and caveolin/raft-mediated endocytosis. Cholesterol might be involved in the pDNA translocation through the plasma membrane. This is the first direct experimental evidence of the occurrence of endocytosis in gene electrotransfer.

Graphical abstractCell permeabilization and formation of the pDNA/membrane interaction due to the application of electric pulses seem to be cholesterol-independent processes, in contrast to gene expression which decreases when cells are treated with the drug MβCD. The pDNA enters the cells via at least two pathways: caveolin/raft-mediated endocytosis (∼50%) and clathrin-mediated endocytosis (∼25%). As endocytotic processes are cholesterol-dependent it may explain why the gene expression is affected by cholesterol depletion.Figure optionsDownload full-size imageDownload as PowerPoint slide